In normal cells, the response to interferony (IFNy) requires the formation of tyrosine-phosphorylated STAT1 dimers for the expression of virtually all genes. However, the work of several laboratories, includingour own, has revealed crucial roles for pathways that must function in addition to the formation of STAT dimers in several different situations. The major focus of this application is to investigate the mechanisms of these novel essential pathways of response to IFNy. Published results from several laboratories show that a variety of kinases are activated in response to IFNy (PIS kinase, AKT, SRC, PKR, IKK and the MAP kinases ERK 1,ERK 2 and p38), along with other intermediate signalingmolecules (adaptors, phosphatases, etc). Furthermore, new evidencefrom our lab reveals that several transcription factors (for example, cMYB) are activated to bind to DNA, as assayed in EMSA analyses, in response to IFNy. We hypothesize that different combinations of STAT1 dimers and other IFNy-activated transcription factors are required to activate individual genes. Aim 1: Novel essential pathways for IFNy-dependent gene expression. We will connect the func- tions of specific phosphotyrosine residues of IFNy receptor subunit 1(IFNGR1) to specific intermediate signaling molecules (kinases, adaptors, etc)to specific novel essential transcription factors to specific genes. Major tools are array-based analysis of IFNy-induced gene expression, use of cells carrying Y-F mutants of IFNGR1, methods to ablate the functions of specific intermediate signaling molecules and transcription factors, methods to analyze transcription factor binding sites (CHIP assays, promoter mutations), and use of specific promoters in genetic experiments. Aim 2: Role of IKK in IFN-dependent signaling. We have found that mouse embryo fibroblasts (MEFs) lacking the two catalytically active IkB kinase (IKK) subunitsare defective in the activation of many genes in response to IFNsp and y. We will study gene expression in MEFs lackingeach of the IKK subunits individually and will use the approaches outlined in Aim 1 to study how these defects affect the activation of intermediate signaling molecules and transcription factors in response to IFNs. We will also use a variety of methods to discover how IKK is activated in response to IFNs. Aim 3: Use novel genetic methods to discover new cellular components required for the full response to IFNy. We will use the IP-10 and GBP promoters, which do not respond to IFNy in IKK-null cells, to drive the expression of selectable markers, exploiting new methods for performinginsertional mutagenesis in mammalian cells. We will use retroviral vectors to generate mutations, followed by reversal of the mutant phenotype when the insertions are excised, and identification of the gene targeted by the insertion. We will also obtain mutant cells that fail to activate a promoter that uses a cMYB binding site in addition to a STATl-tropic GAS site to drive gene expression. Results of this approach will set the stage for further investigation of how these pathways help to determine the biological responses to the IFNs.